1. Field of the Invention
The present invention relates to gene expression, including regulation, in mammalian cell and transgenic animal systems.
2. Discussion of the Background
Although many characterized structural genes, together with control sequences, have been inserted into vectors and used to transform host cells, there remains a need for expression vectors capable of exhibiting high levels of expression in varied systems, and which preferably can be regulated. In particular, expression vectors for use in either mammalian cell lines or transgenic systems, possessing these characteristics are becoming increasingly important, both for the commercial production of desired polypeptides and for the development of therapies and/or treatments for diseases and genetic disorders.
The level of gene expression obtainable in transgenic animals, to date, has often been very low and sometimes undetectable. The reasons for this low expression are poorly understood, but may relate to inefficient activation of gene expression during development. In general, intact genes with sufficient flanking sequences are expressed best, chimeric genes made of heterologous promoters and intact structural genes are expressed quite reliably, whereas cDNA construct with heterologous promoters are the most troublesome.
Some of the inventors have been trying to find a general method for expressing cDNA constructs in transgenic mice (Brinster et al, Proc. Nat. Acad. Sci. USA (1988) 85: 836-840; Palmiter et al, Proc. Nat. Acad. Sci. USA (1991) 88:478-482. Because cDNA lacks introns, the inventors initially thought that adding introns to these constructs might improve expression, and it does in some situations (Palmiter et al, 1991). Although one generally thinks of introns as having a major role in RNA splicing, the inventors believe that introns affect gene expression at the transcriptional level, perhaps by allowing favorable phasing of nucleosomes relative to important cis-acting promoter elements.
When making transgenic animals, e.g. transgenic mice, by the microinjection method, the transgenes are thought to integrate randomly into the genome and they usually integrate in tandem, head-to-tail arrays. Although the genes may be expressed from many random integration sites, the level of expression in, for example, different mouse lines varies tremendously and is usually unrelated to transgene copy number. The general explanation to this phenomena is that the transgenes are subject to local chromosomal effects, called "position effects".
It is thought that genes may normally reside in chromosomal domains that insulate them from neighboring chromosomal effects. Experimental support for this latter suggestion comes from the work on the globin locus in which sequences flanking the locus, now called locus control regions (LCR), can convey position-independency and copy number-dependency to globin genes (Grosveld et al, Cell (1987) 51:975-985) and to heterologous genes. However, the LCR from the globin gene only functions in erythroid cells and its use is thus very limited. Being interested in a general solution to the gene expression problem, the inventors have sought sequences that have a similar property but are capable of functioning in many, if not all, cell types.
Metallothionein (MT) genes encode small proteins that bind heavy metals such as zinc, cadmium and copper by virtue of their high cysteine content. These genes have been found in all eukaryotic organisms examined and they are thought to play an important role in metal homeostasis and resistance to metal toxicity (for a review see: Kagi et al, Experientiia Supplementum (1987), 52, Birkhauser Verlag, Basel). In most mammals there are at least two similar MT genes, MT-I and MT-II, that as far as is known serve identical functions. In the mouse, these genes are closely linked on chromosome 8 and they are coordinately regulated by metals, glucocorticoids and acute-phase stimulators (Searle et al, Mol. Cell. Biol. (1984) 4:1221-1230).
Many of the cis-acting elements that mediate the transcriptional responses to these stimuli have been located to within a few hundred base pairs upstream of the transcription start site (for review see: Hamer, Annu. Rev. Biochem. (1986) 55:913-951). MT genes are expressed in various cell types throughout mouse development and in most cells of the adult; their regulation in vivo is a complex function of cellular exposure to stimuli and the presence of appropriate signal transduction pathways (Searle et al, Mol. Cell. Biol. (1984) 4: 1221-1230; De et al, J. Biol. Chem. (1990) 265: 15267-15274).
The metallothionein (MT) promoter has been used to direct expression of a wide variety of reporter genes in transgenic mice (Palmiter et al, Ann. Rev. Gen. (1986) 20: 465-499). In general, one observes expression of the reporter gene in those tissues that normally express MT well, such as liver, kidney and intestine, and the expression is often inducible by heavy metals. However, the expression pattern of the reporter gene and its responsiveness to various stimuli rarely mimics that of the endogenous MT genes exactly and there are often bizarre exceptions such as expression in one tissue only. In addition, the level of expression is usually unrelated to transgene copy number (Palmiter et al, 1986).
These results suggest that while certain control elements may be functional, the expression of MT transgenes is strongly influenced by the site of chromosomal integration. All of these experiments are complicated by the fact that the reporter gene being used in conjunction with the MT promoter may markedly influence the expression pattern by affecting transcription and/or mRNA stability, and these effects may not parallel those of endogenous MT genes in all tissues.
Similar results have been observed when the DNA sequences which regulate the human .beta.-globin gene were introduced 5' and 3' to various cloned globin genes (Charnay et al, Cell (1984) 38: 251-263; Wright et al, Nature (1983) 305: 333-336). When a .beta.-globin gene containing these DNA sequences were introduced into mice, the gene was not expressed at the same level as the mouse .beta.-globin gene and exhibited integration site dependent effects (Townes et al, EMBO J. (1985) 7: 1715. This was characterized by a highly variable expression of the transgene that did not correlate with the copy number of the injected gene in the mouse genome.
As noted above, Grosveld et al have demonstrated that when the human .beta.-globin gene with its own promoter and enhancers ligated to DNA sequences that lie upstream of .epsilon.-globin gene and shown to be hypersensitive to DNase digestion, the expression pattern obtained in transgenic mice is position-independent and directly correlates to the number of inserted copies. However, the effect of these flanking regions is cell-specific and thus of limited use. The expression level per gene copy is at a similar level to that of endogenous globin genes in erythroid cells. In non-erythroid cells, low levels of expression are obtained, similar to those obtained when the flanking regions are not present (Blom van Assendelft et al, Cell (1989) 56: 969-977).
In a chromosome, the genetic material is packaged into a DNA/protein complex called chromatin which has the effect of limiting the availability of DNA for functional purposes. It has been established that many gene systems possess so-called DNA hypersensitive sites. Such sites representative putative regulatory regions, where the normal chromatin structure is altered by binding of proteins to specific DNA sequences. For example, DNaseI hypersensitive sites are often associated with the promoter and enhancer regions of active genes. The presence of DNaseI hypersensitive sites (in the vicinity of genes) that are not directly related to gene expression, suggests that they may mark the location of other important chromosomal functions, perhaps boundaries of chromosomal domains, origins of replication, or sites of attachment to nuclear matrix.
MacArthur et al (J. Biol. Chem. (1987) 7: 3466-3472) mapped DNaseI hypersensitive sites in a thymoma-derived cell line, S49. These cells are unusual in that neither of the MT genes are expressed nor inducible by metals. However, cadmium-resistant variants can be selected that express either MT-I, MT-II or both genes. DNaseI hypersensitive sites flanking the MT genes (about 6 kb 5' of the MT-II gene and 4 kb 3' of the MT-I gene) are present regardless of whether the MT genes are expressed, but new hypersensitive sites appear in the promoter regions of whichever MT genes are expressed after selection (MacArthur et al, 1987).
Several other genetic loci are also flanked by DNaseI hypersensitive sites, including the human .beta.-globin locus (Tuan et al, Proc. Nat. Acad. Sci. U.S.A., (1985) 82: 6384-6388; Proc. Nat. Acad. Sci. U.S.A. (1986) 83: 1359-1363) and the chicken lysozyme gene (Sippel et al, Nucleic Acids and Molecular Biology 3, Springer Verlag: Berlin, 1988, pp. 1323-147).
Pruzina et al (Nucleic Acid Research (1991) 19: 1413-1419) defines an LCR of the human .beta.-globin gene domain located upstream of the human .beta.-globin multigene cluster and divided into four DNaseI hypersensitive sites (HS). The LCR HSS4 has been precisely mapped to a 280 bp fragment that has functional LCR activity in MEL cells and transgenic mice. The sequence of the 280 bp fragment is disclosed in this publication.
Philipsen et al (EMBO J. (1990) 9: 2159-2167) describes the hypersensitive site 2 (HS2) of the LCR of the human .beta.-globin gene. The publication discloses a 225 bp fragment sufficient to direct high levels of expression of the human .beta.-globin gene in a copy number dependent and integration site independent fashion. The sequence of the 225 bp fragment is also disclosed.
Talbot et al (Nature (1989) 338: 352-355) defines the human .beta.-globin LCR region properties as described above. It is located in a 6.25 kb section of DNA found 5' of the human .beta.-globin locus. This region allows high levels of expression of the human .beta.-globin gene as well as a heterologous thymidine kinase gene in erythroid cells of transgenic mice.
Bonifer et al (EMBO J. (1990) 9: 2843-2848), discloses transgenic mice that carry the entire wild type chicken lysozyme gene domain including the 11.5 kilobase 5' flanking and 5.53 sequences (LCR or A-elements). The publication reports the cross species gene transfer ability of chicken genes in mammals resulting in high level macrophage-specific gene expression in the recipient mouse, and that the entire gene locus transferred into the mouse acts as an independent regulatory unit not requiring a specific chromosome position.
Steif et al (Nature, (1989) 341: 343-345) describes how a reporter gene encoding CAT flanked by 5' A-elements (LCRs) from the chicken lysozyme gene may have significant elevated expression in stably transfected cells and that the expression is independent of the chromosome position.
Reitman et al (Nature (1990) 348: 749-752) reports studies of chicken .beta.-globin gene expression in transgenic mice. Chicken DNA elements that reportedly allow position independent expression that can function in mice are described. These elements are within 2 kb from the chicken globin gene demonstrating an intracluster location that differs from the LCR location found in human .beta.-globin and chicken lysozyme.
Greaves et al (Cell (1989) 56: 979-986) describes LCR sequences located downstream from the human CD2 gene that activates high level T-cell specific expression in transgenic mice regardless of the site of the chromosome integration of the transgene.
The following publications are also of interest: Albitar et al, Mol. Cell. Bio. (1991) 11: 3786-3794; Constantoulakis et al, Blood (1991) 77: 1326-1333; Kulocik et al, J. Clin Invest. (1991) 87: 2142-2146; Talbot et al, EMBO J. (1991) 10: 1391-1398; Dillon et al, Nature (1991) 350: 352-354; Hanscombe et al, Genes and Devel. (1991) 5: 1387-1394; Catarina et al, Proc. Natl. Acad. Sci. (U.S.A.) (1990) 88: 1626-1630; Lavelle et al, Proc. Natl. Acad. Sci. U.S.A. (1990) 88: 7318-7322; Raich et al, Science (1990) 250: 1147-1149; Greaves et al, Nature (1990) 343: 183-185; Orkin, Cell (1990) 63: 665-672; Grosveld, Ann. New York Acad. of Sci. (1990) 6112: 152-159; Palmiter et al, Ann. Rev. Genet. (1986) 20: 465-499; Townes et al, EMBO J. (1985) 4: 1715-1723; Muller et al, Bone Marrow Transplantation (1990) 5: 13-14; Peters et al, Eur. J. Biochem. (1989) 182: 507-516; Fritton et al, Biol. Chem. Hoppe-Seyler (1987) 368: 111-119; and Theisen et al, EMBO J. (1986) 5: 719-724.
The sequences including the DNaseI hypersensitive sites confer copy number-dependent and position-independent expression upon transgenes (Grosveld et al, 1987; Townes et al, Trends Genetics (1990) 6: 219-223; Bonifer et al, EMBO J. (1990) 9: 2843-2848). The mechanism of action of these sequences, or locus control regions (LCR), is not well defined, but they may help establish a chromosomal domain that facilitates transcription of genes from adjacent chromosomal effects (Elgin, New Biologist (1991) 3: 37-42; Felsenfeld, Nature (1992) 355: 219-224).
No system has been described however which provides tissue-independent, high-level, copy-number dependent, position-independent expression in mammalian cells and/or transgenic systems, much less such a system which is susceptible to regulation. As a matter of fact, no evidence has been reported that such a system exists. Only the existence of cell type-restricted expression sequences associated with gene clusters involved in developmental differentiation have been reported, e.g. the human .beta.-globin gene cluster, 5'-.epsilon.-.sup.G .gamma.-.sup.A .gamma.-.delta.-.beta.-3' described by Grosveld et al (WO 89/01517).